Biomarker-based histological work is known to save lives, help the formulation of therapeutic intervention strategies, and allow for improved prognosis (See, Nathwani B. N. et al., Adv. Anat. Pathol., 2007, 14, 375-400 and J. Teruya-Feldstein, Arch. Pathol. Lab. Med., 2010, 134, 1659-1665). Among all the known biomarkers, cancer cell surface carbohydrate antigens play a very important role, and most clinically measured cancer biomarkers are glycoproteins (See, Ludwig J. A. et al., Nat. Rev. Cancer, 2005, 5, 845-856). Cell surface carbohydrate structures as part of glycosylated proteins, peptides, and lipids are characteristic signatures of different cell types and are associated with many forms of cancer. For example, the sialyl Lewis X (sLex) antigen is being assessed in many cancers; serum sLex and cytokeratin 19 fragment are said to be predictive factors for recurrence in patients with stage I non-small cell lung cancer; and sLex plus CA 15.3 levels in breast cancer serum were reported to be more effective than CA 15.3 plus CEA (See, Mizuquchi S. et al., E. J. Cancer Suppl., 2007, 6554 and Kurebayashi J. et al., Jpn. J. Clin. Oncol., 2006, 36, 150-153). Furthermore, the combination of sLex and sLea expression has been shown to mediate adhesion of urothelial cancer cells to activated endothelium. Detection of the changes in expression of these cell surface carbohydrates is clearly very important in cancer histological work.
The folate receptor, a tumor associated glycosylphosphatidylinositol anchored protein, is upregulated in more than 90% of non-mucinous ovarian carcinomas. It is also found at high to moderate levels in kidney, brain, lung, and breast carcinomas while it occurs at very low levels in most normal tissues (Kamen B. A., et al., “A Review of Folate Receptor Alpha Cycling and 5-Methyltetrahydrofolate Accumulation with an Emphasis on Cell Models in vitro,” Adv. Drug Delivery Rev. 2004, 56 1085-1097). The folate receptor density also appears to increase as the stage of the cancer increases (Elnakat, H., et al., “Distribution, Functionality and Gene Regulation of Folate Receptor Isoforms: Implication in Targeted Therapy,” Adv. Drug Delivery Rev. 2004, 56 1067-1084).
Tumor cells are characterized by uncontrolled growth, invasion to surrounding tissues, and metastatic spread to distant sites. Mortality from cancer is often due to metastasis since surgical removal of tumors can enhance and prolong survival. The integrins constitute a family of transmembrane receptor proteins composed of heterodimeric complexes of noncovalently linked alpha and beta chains. Integrins function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions and transduce signals from the ECM to the cell interior and vice versa. Hence, the integrins mediate the ECM influence on cell growth and differentiation. Since these properties implicate integrin involvement in cell migration, invasion, intra- and extra-vasation, and platelet interaction, a role for integrins in tumor growth and metastasis has been established. These findings are underpinned by observations that the integrins are linked to the actin cytoskeleton involving talin, vinculin, and alpha-actinin as intermediaries. Such cytoskeletal changes can be manifested by rounded cell morphology, which is often coincident with tumor transformation via decreased or increased integrin expression patterns. For the various types of cancers, different changes in integrin expression are further associated with tumor growth and metastasis. Tumor progression leading to metastasis appears to involve equipping cancer cells with the appropriate adhesive (integrin) phenotype for interaction with the ECM. Therapies directed at influencing integrin cell expression and function are presently being explored for inhibition of tumor growth, metastasis, and angiogenesis. Such therapeutic strategies include anti-integrin monoclonal antibodies, peptidic inhibitors (cyclic and linear), calcium-binding protein antagonists, proline analogs, apoptosis promotors, and antisense oligonucleotides. Moreover, platelet aggregation induced by tumor cells, which facilitates metastatic spread, can be inhibited by the disintegrins, a family of viper venom-like peptides. Therefore, adhesion molecules from the integrin family and components of angiogenesis might be useful as tumor progression markers for prognostic and for diagnostic purposes. Development of integrin cell expression profiles for individual tumors may have further potential in identifying a cell surface signature for a specific tumor type and/or stage. Thus, recent advances in elucidating the structure, function, ECM binding, and signaling pathways of the integrins have led to new and exciting modalities for cancer therapeutics and diagnoses.
In histological work, fluorescent and/or color staining agents are most commonly used. This approach, however, suffers from difficulties in multiplexing due to spectral resolution/overlap issues and in quantitation. A novel but maturing technology, MALDI imaging mass spectrometry (MALDI-IMS) allows for direct examination of tissue biopsies without the need for micro-dissection and solubilization of tissue biomarkers prior to analysis, and ion desorption can be targeted to specific “points” in a grid pattern and the data rasterized. The resulting spectra can then be used to generate two-dimensional molecular maps of hundreds of biomolecules directly from the surface of a tissue section. These molecular maps display the relative abundance and spatial distribution of these molecules. MALDI tissue profiling has the power to link the molecular detail of mass spectrometry with molecular histology, generating mass spectra correlated to known locations within a thin tissue section. We and others have recently demonstrated the potential of MALDI-IMS to clinical histopathology applications.
There is therefore a long felt need for techniques using cell surface specific agents that can be used for identifying cancerous tissue and therefore the need for cell surface specific biomarkers.